The cytosolic space of cells is an important target for drug delivery systems since many therapeutic agents are aimed at intervening or modifying specific molecular events occurring in the cytoplasm of cells. Many of these therapeutic agents, particularly the ones discovered or designed in recent years, are intrinsically membrane-permeant due to their charge or high molecular weight. The long term objective of this proposal is to develop strategies and methods that can deliver molecules in this category into the cytosolic space of cells. The main idea of this proposal is to import cell biology and biochemistry of Listeria into pharmaceutics, i.e., to take a minimal component from a facultative intracellular bacterium, Listeria monocytogenes, and construct a liposome-based cytosolic delivery vehicle. Listeriolysin O (LLO), which is necessary and sufficient in the escape of Listeria into cytosol, will be purified and incorporated into liposomes. The LLO-containing liposomes, which mimic how Listeria deliver themselves into the cytosol where they grow, will be characterized in terms of their ability to delivery macromolecules into the cytosol. Although the proposed strategy should be applicable to the delivery of other types of macromolecules, the current proposal will focus on the delivery of proteinaceous macromolecules. Two types of proteins that show effect when delivered into the cytosol will be tested: (1) an antigenic protein that induces activation of specific cytotoxic T lymphocytes (CTL); and (2) toxins that can inhibit protein synthesis. The amount of cytosolic delivery will be measured by a newly developed biochemical assay which monitors the percent of cytosolically delivered molecules out of the total cell-associated amount. In vitro antigen presentation assays will also be employed to measure relative efficiency of delivery into the cytosolic pathway of antigen presentation. This has direct implications as a vaccine delivery vehicle, and will therefore be extended to a well-established mouse model of lymphocytic choriomeningitis virus (LCMV) infection. Induction of CTL, specific against nucleoprotein (NP) of LCMV, by NP-containing LLO-liposomes and subsequent protection from viral challenge will be monitored in mice. In addition to delivering antigenic proteins, a membrane-impermeant toxin, gelonin, will be encapsulated inside LLO-liposomes and tested in the cell growth inhibition assays. The liposome formations that have a long circulation time will be made with LLO, and their efficiency of cytosolic delivery will also be tested. Concurrently, the mechanism of endosome lysis by LLO via pore formation will be studied using biophysical and molecular biological methods.